Project Summary This project is focused on feasibility testing of a new device based on electromechanical signal transduction for the low-cost (~$10 in electronics), optics-free and amplification-free (e.g., no PCR) detection of DNA/RNA at ultralow concentration. The device addresses the projected $12.8B disease diagnostics market and is well suited for the detection of bacterial pathogens in body fluids, food and water through the presence of specific 16S rRNA sequences. A compelling need persists for rapid (minutes), cost effective, point-of-care (POC) nucleic acid (NA) detection devices for infectious disease diagnostics. The investigators recently demonstrated detection of Escherichia coli (E. coli) 16S rRNA against a 106-fold background of Pseudomonas putida RNA at <10-18 M (<1 CFU/10 mL). This ultralow detection limit was achieved using a simple RNA extraction and hybridization protocol that is accomplished in <30 mins at the lab bench. It is likely that an integrated microfluidic device eventually can be produced for pathogen detection in <15 mins. A key feature of the proposed device is the use of peptide nucleic acid (PNA) capture probes, which are uncharged polyamide analogs to NAs that share the same base chemistry. Since the bead-PNA conjugates are designed to be charge neutral, they do not exhibit electrophoretic movement in the presence of a DC electric field. However, the substantial negative charge acquired upon capture of a target NA sequence makes the hybridized conjugate mobile. Electrophoresis of the bead-PNA conjugate with hybridized target NA to the mouth of a smaller diameter glass pore causes a significant increase in pore resistance, thereby resulting in a strong, sustained drop in measured ionic current. Nonspecifically bound NA is removed from the bead conjugate in the strong electric field in the pore mouth resulting in no sustained signal. Further, the opposing electroosmotic flow through the glass pore sweeps bead-PNA conjugates without hybridized target away from the pore mouth. In such a way, this simple conductometric device gives a highly selective, binary response signaling the presence or absence of the target NA (and associated pathogen). This project focuses on 2 Aims: 1) Demonstration of selective detection of E. coli in human urine against a background of normal flora at a statistical performance level competitive or better than existing commercial devices and 2) Development and testing of the inexpensive, device-associated electronics that are suitable for manufacturing. Successful achievement of these Aims will substantiate the potential of the novel device for integration into a microfluidic system for detection of Neisseria gonorrhoeae, a CDC top-three public health threat, and Chlamydia trachomatis in human urine. Further, this project will illustrate the potential of the technology for development of generally applicable, inexpensive POC devices for bacterial pathogens in body fluids, food and water.